ABSTRACT The goal of this CETR is to use a combination of innovative strategies to combat drug resistant infectious diseases. In particular, we will use cutting edge methods in metabolomics and genomics to discover lead small molecules targeting the most problematic multi-drug resistant (MDR) Gram-negative bacterial and fungal pathogens. This project, Project 2 (P2), will develop and implement a metabolomics pipeline that will also serve Project 1 and Project 3. We will integrate bioassay data from multiple pathogens exhibiting diverse drug resistant phenotypes to supplement our metabolomics data. Essentially, we will employ metabolomics-driven pipeline to identify novel molecules with target efficacy both in vitro and in vivo againswhile also lacking host toxicity traits. To identify and access chemical diversities that will drive this project, we will target symbiotic microbes associated with i) marine invertebrates, and ii) insects; in all cases a particular focus will be placed on rare bacterial genera. Additionally, we seek answers to basic questions underpinning these new strategies and their high success rates relative to historical discovery approaches. To answer these questions and discover the next generation of antimicrobials, projects P1, P2 and P3 share three identical specific aims; Aims 1-3 all focus on how symbioses provide novel bacteria as sources for new compound discovery with in vivo efficacy against MDR pathogens. Support for this project will be supplied by four scientific cores (Chemistry, In Vitro, In Vivo, and Mechanism of Action). The outcome of this project will be novel antimicrobial small molecule leads with in vivo efficacy against MDR pathogens and host safety.